Naoko Yoshida

Phylogenetic characterization of a polychlorinated-dioxin- dechlorinating microbial community by use of microcosm studies.

ABSTRACT Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.

A novel Dehalobacter species is involved in extensive 4,5,6,7-tetrachlorophthalide dechlorination.

ABSTRACT The purpose of this study was the enrichment and phylogenetic identification of bacteria that dechlorinate 4,5,6,7-tetrachlorophthalide (commercially designated “fthalide”), an effective fungicide for rice blast disease. Sequential transfer culture of a paddy soil with lactate and fthalide produced a soil-free enrichment culture (designated the “KFL culture”) that dechlorinated fthalide by using hydrogen, which is produced from lactate. Phylogenetic analysis based on 16S rRNA genes revealed the dominance of two novel phylotypes of the genus Dehalobacter (FTH1 and FTH2) in the KFL culture. FTH1 and FTH2 disappeared during culture transfer in medium without fthalide and increased in abundance with the dechlorination of fthalide, indicating their growth dependence on the dechlorination of fthalide. Dehalobacter restrictus TEA is their closest relative, with 97.5% and 97.3% 16S rRNA gene similarities to FTH1 and FTH2, respectively.

Bmi1 expression in long-term germ stem cells

Asingle cells in undifferentiated spermatogonia are considered to be the most primitive forms of germ stem cells (GSCs). Although GFRα1 is thought to be a marker of Asingle cells, we found that Bmi1High is more specific than GFRα1 for Asingle cells. Bmi1High expression in Asingle cells is correlated with seminiferous stages, and its expression was followed by the proliferative stage of Asingle GSCs. In contrast, GFRα1 expression was seminiferous stage-independent. Fate analyses of EdU-positive Bmi1High-positive cell-derived Asingle cells revealed that these cells self-renewed or generated transient amplifying Apaired cells. Bmi1High-positive cells were resistant to irradiation-induced injury, after which they regenerated. Elimination of Bmi1High-positive cells from seminiferous tubules resulted in the appearance of tubules with seminiferous stage mismatches. Thus, in this study, we found that Bmi1High is a seminiferous stage-dependent marker for long-term GSCs and that Bmi1High-positive cells play important roles in maintaining GSCs and in regenerating spermatogenic progenitors after injury.

Anaerobic mineralization of pentachlorophenol (PCP) by combining PCP-dechlorinating and phenol-degrading cultures.

The dechlorination and mineralization of pentachlorophenol (PCP) was investigated by simultaneously or sequentially combining two different anaerobic microbial populations, a PCP‐dechlorinating culture capable of the reductive dechlorination of PCP to phenol and phenol‐ degrading cultures able to mineralize phenol under sulfate‐ or iron‐reducing conditions. In the simultaneously combined mixture, PCP (about 35 µM) was mostly dechlorinated to phenol after incubation for 17 days under sulfate‐reducing conditions or for 22 days under iron‐reducing conditions. Thereafter, the complete removal of phenol occurred within 40 days under both conditions. In the sequentially combined mixture, most of the phenol, the end product of PCP dechlorination, was degraded within 12 days of inoculation with the phenol degrader, without a lag phase, under both sulfate‐ and iron‐reducing conditions. In a radioactivity experiment, [14C–U]–PCP was mineralized to 14CO2 and 14CH4 by the combined anaerobic microbial activities. Analysis of electron donor and acceptor utilization and of the production and consumption of H2, CO2, and CH4 suggested that the dechlorinating and degrading microorganisms compete with other microorganisms to perform PCP dechlorination and part of the phenol degradation in complex anoxic environments in the presence of electron donors and acceptors. The presence of a small amount of autoclaved soil slurry in the medium was possibly another advantageous factor in the successful dechlorination and mineralization of PCP by the combined mixtures. This anaerobic–anaerobic combination technology holds great promise as a cost‐effective strategy for complete PCP bioremediation in situ. Biotechnol. Bioeng. 2009;102: 81–90.

A humin-dependent Dehalobacter species is involved in reductive debromination of tetrabromobisphenol A

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant on the market. It has been detected in various environmental samples, and a growing body of evidence has demonstrated its toxic effects on living organisms. In this study, we report the enrichment and phylogenetic identification of bacteria that debrominate TBBPA to bisphenol A in the presence of humin. Incubation experiments indicated that humin was required for this debromination activity. Of the five compounds examined for inclusion in the TBBPA-debrominating culture, formate was the optimal electron donor. A 16S rRNA gene library showed that the culture was dominated by three known dehalogenator genera: Dehalobacter, Geobacter, and Sulfurospirillum. Further investigation indicated that Dehalobacter was responsible for the debromination of TBBPA. PCR-denaturing gradient gel electrophoresis analysis showed that Dehalobacter grew in the culture by utilizing TBBPA. Moreover, the copy number of the Dehalobacter 16S rRNA genes increased by about two orders of magnitude in the cultures without the addition of TBBPA, whereas it increased by approximately four orders of magnitude when TBBPA was present. The incubation experiments showed that Dehalobacter was reliant on humin for its debromination activity, indicating a new type of metabolism in Dehalobacter that is linked to humin respiration.

Complete anaerobic mineralization of pentachlorophenol (PCP) under continuous flow conditions by sequential combination of PCP-dechlorinating and phenol-degrading consortia

Complete mineralization of 50 µM of pentachlorophenol (PCP) was achieved anaerobically under continuous flow conditions using two columns connected in series with a hydraulic retention time of 14.2 days, showing the highest reported mineralization rate yet of 3.5 µM day−1. The first column, when injected with a reductive PCP dechlorinating consortium, dechlorinated PCP to mainly phenol and traces of 3‐chlorophenol (3‐CP) using lactate supplied continuously as an electron donor. The second column, with an anaerobic phenol degrading consortium, decomposed phenol and 3‐CP under iron‐reducing conditions with substantial fermentative degradation of organic compounds. When 20 mM of lactate was introduced into the first column, the phenol degradation activity of the second column was lost in a short period of time, because the amorphous Fe(III) oxide (FeOOH) that had been packed in the column before use was depleted by lactate metabolites, such as acetate and propionate, flowing into the second column from the first column. The complete mineralization of PCP was maintained for a long period by reducing the lactate concentration to 4 mM, effectively extending the longevity of second‐column activity with no depletion of FeOOH for more than 200 pore volumes (corresponding to 3,000 days). The carbon balance showed that 50 µM PCP and 4 mM lactate in the influent had transformed to CO2 (81%) and CH4 (3%) and had contributed to biomass growth (8%). A comparison of the microbial consortia introduced into the columns and those flowing out from the columns suggested that the introduced population did not flow out during the experiments, although the microbial composition of the phenol column was considered to be affected by the inflow of microbes from the PCP dechlorination column. These results suggest that a sequential combination of reductive dechlorinating and anaerobic oxidizing consortia is useful for anaerobic remediation of chlorinated aromatic compounds in a microbial permeable reactive barrier. Biotechnol. Bioeng. 2010;107: 775–785.

Reductive dechlorination of chloroethenes by Dehalococcoides-containing cultures enriched from a polychlorinated-dioxin- contaminated microcosm

The reductive dechlorinating abilities for chloroethenes of seven enrichment cultures from polychlorinated-dioxin-dechlorinating microcosm were investigated using culture-independent and -dependent methods. These cultures were constructed and maintained with 1,2,3-trichlorobenzene (1,2,3-TCB) or fthalide as an electron acceptor and hydrogen as an electron donor. Denaturing gradient gel electrophoresis (DGGE) analysis of the amplified fragments targeting the 16S rRNA gene showed one or two major bands, whose nucleotide sequences were then analyzed and were found to suggest that Dehalococcoides was one of the dominant bacteria in all enrichment cultures. The nucleotide sequence data revealed that the identity of the major band was 100% identical to the 16S rRNA gene sequence of the Pinellas subgroup of the Dehalococcoides clusters, that is, strains CBDB1 and FL2. Genetic diagnosis targeting the pceA, tceA, bvcA, vcrA and reductive dehalogenase homologous (rdh) gene was performed to investigate the potential for reductive chloroethene dechlorination of cultures. The required length of PCR-amplified fragments was not observed, suggesting that these cultures are not capable of reductively dechlorinating chloroethenes. However, a culture-dependent test indicated that two cultures, TUT1903 and TUT1952, reductively dechlorinated tetrachloroethene (PCE) to trichloroethene (TCE), although not completely. While, TUT2260 and TUT2264 completely converted PCE to TCE and dichloroethenes, but not further. These results suggest that these TUT cultures might include a novel type of bacteria belonging to the Dehalococcoides group and that currently available information on both the 16S rRNA gene and rdh gene sequences is insufficient to definitively evaluate the potential abilities for reductive dechlorination.

Isolation and Functional Gene Analyses of Aromatic-Hydrocarbon-Degrading Bacteria from a Polychlorinated-Dioxin-Dechlorinating Process

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 105 copies of the AhDO large subunit genes g−1 wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from “Dehalococcoides” and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

Aquatic plant surface as a niche for methanotrophs

This study investigated the potential local CH4 sink in various plant parts as a boundary environment of CH4 emission and consumption. By comparing CH4 consumption activities in cultures inoculated with parts from 39 plant species, we observed significantly higher consumption of CH4 associated with aquatic plants than other emergent plant parts such as woody plant leaves, macrophytic marine algae, and sea grass. In situ activity of CH4 consumption by methanotrophs associated with different species of aquatic plants was in the range of 3.7–37 μmol·h−1·g−1 dry weight, which was ca 5.7–370-fold higher than epiphytic CH4 consumption in submerged parts of emergent plants. The qPCR-estimated copy numbers of the particulate methane monooxygenase-encoding gene pmoA were variable among the aquatic plants and ranged in the order of 105–107 copies·g−1 dry weight, which correlated with the observed CH4 consumption activities. Phylogenetic identification of methanotrophs on aquatic plants based on the pmoA sequence analysis revealed a predominance of diverse gammaproteobacterial type-I methanotrophs, including a phylotype of a possible plant-associated methanotroph with the closest identity (86–89%) to Methylocaldum gracile.

Anaerobic mineralization of 2,4,6-tribromophenol to CO2 by a synthetic microbial community comprising Clostridium, Dehalobacter, and Desulfatiglans

Anaerobic mineralization of 2,4,6-tribromophenol (2,4,6-TBP) was achieved by a synthetic anaerobe community comprising a highly enriched culture of Dehalobacter sp. phylotype FTH1 acting as a reductive debrominator; Clostridium sp. strain Ma13 acting as a hydrogen supplier via glucose fermentation; and a novel 4-chlorophenol-degrading anaerobe, Desulfatiglans parachlorophenolica strain DS. 2,4,6-TBP was debrominated to phenol by the combined action of Ma13 and FTH1, then mineralized into CO2 by sequential introduction of DS, confirmed using [ring-(14)C(U)] phenol. The optimum concentrations of glucose, SO4(2-), and inoculum densities were 0.5 or 2.5mM, 1.0 or 2.5mM, and the densities equivalent to 10(4)copiesmL(-1) of the 16S rRNA genes, respectively. This resulted in the complete mineralization of 23μM 2,4,6-TBP within 35days (0.58μmolL(-1)d(-1)). Thus, using a synthetic microbial community of isolates or highly enriched cultures would be an efficient, optimizable, low-cost strategy for anaerobic bioremediation of halogenated aromatics.